Seed Train Optimization in Microcarrier-Based Cell Culture Post In Situ Cell Detachment through Scale-Down Hybrid Modeling.

Microcarrier-based cell culture is a commonly used method to facilitate the growth of anchorage-dependent cells like MA 104 for antigen manufacturing. However, conventionally, static cell culture is employed for cell propagation before seeding the production bioreactor with microcarriers (MCs). This study demonstrates the effective replacement of the conventional method by serial subculturing on MCs with in situ cell detachment under optimal conditions in closed culture units. This study proves that MA 104 can be subcultured at least five times on Cytodex 1 MC without the need for separating cells and MC after cell harvest. Process parameters impacting cell growth were studied post in situ cell detachment in a scaled-down model. Optimization, using augmented Design of Experiments (DoE) combined with hybrid modeling, facilitated rapid screening of the design space for critical process parameters (CPPs). Optimized conditions included an inoculation density of >16 cells/bead, 3.5-4.5 g/L of Cytodex 1, and a controlled agitation speed, starting at Njs (minimum agitation speed) for the first day with a maximum increase of 25% thereafter. With these design spaces for CPPs, a cell density of 2.6 ± 0.5 × 106 cells/mL was achieved after five days. This refined bioprocess methodology offers a reliable and efficient approach for seed training in stirred tank reactors, which is particularly beneficial for viral vaccine production.

Effects of Blood-Derived Products on Cellular Senescence and Inflammatory Response: A Study on Skin Rejuvenation.

Blood-derived products, such as citrate platelet-rich plasma (CPRP) and hyperacute serum (HAS), are recognized for their rich growth factor content. When human dermal fibroblast (HDF) cells are exposed to combined mitogenic and DNA-damaging stimuli, it can lead to an increased burden of senescent cells and a modified senescence-associated secretory phenotype. In this study, the senescent state was comprehensively assessed through various methods, including phosphorylated histone H2AX (γH2AX) staining, p21 and p16 q-PCR, p21-western blot, growth curves, and senescence-associated ß-galactosidase staining. Two primary treatments with blood products were administered, one early (immediately after etoposide) and the other late (11 days after etoposide treatment). The effects of the blood product treatment were evaluated by measuring interleukin 6 and 8 (IL-6 and IL-8) levels, as well as collagen 1 (COL1) and p21 mRNA expression. Additionally, 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) assays, cell size measurements, viability assays, and cell number calculations were conducted. The results revealed that cells treated with hyperacute serum in the early treatment phase exhibited the lowest observed IL-6 and IL-8 levels. In contrast, a clear inflammatory response for IL-8 was observed in cells treated with hyperacute serum and citrate platelet-rich plasma during the late treatment. Furthermore, an upregulation of COL1 expression was observed in the early treatment, while cells in the late treatment group remained unaffected. Notably, citrate platelet-rich plasma-treated cells showed a decrease in COL1 expression. Overall, the treatment with blood products appears to have slightly positive effects on skin rejuvenation.

Two-Layer Inkjet-Printed Microwave Split-Ring Resonators for Detecting Analyte Binding to the Gold Surface.

This work focuses on demonstrating the working principle of inkjet-printed Au nanoparticle (NP) two-layer Gigahertz (2.6 GHz) microwave split-ring resonators (SRRs) as a novel platform for the detection of analytes on flexible substrates. In contrast to the standard fabrication of split-ring resonator biosensors using printed circuit board technology, which results in a seven-layer system, the resonators in this work were fabricated using a two-layer system. A ground plane is embedded in the SRR measurement setup. In this method, a microwave electromagnetic wave is coupled into the Au SRR via an inkjet-printed Cu-NP stripline that is photonically sintered. This coupling mechanism facilitates the detection of analytes by inducing resonance shifts in the SRR. In this study, the functionality of the printed sensors was demonstrated using two different Au functionalization processes, firstly, with HS-PEG7500-COOH, and, secondly, with protein G with an N-terminal cysteine residue. The sensing capabilities of the printed structures are shown by the attachment of biomolecules to the SRR and the measurement of the resulting resonance shift. The experiments show a clear shift of the resonance frequency in the range of 20-30 MHz for both approaches. These results demonstrate the functionality of the simplified printed two-layer microwave split-ring resonator for use as a biosensor.

IGF1 inclusion bodies: A QbD based process approach for efficient USP as well as early DSP unit operations.

E. coli is an attractive host organism for strong recombinant protein expression. It expresses products either as soluble protein or as inclusion bodies (IB). IBs are insoluble, mostly inactive aggregates. However, recent progress enabled the efficient refolding back into their bioactive structure. Targeted IB production processes have been designed based on their characteristic features such as high yields along with purity and their simple separation. More profound process knowledge is needed to reveal interacting parameters required for quality by design grounded process development. This enables strategies for simplifying and accelerating upstream as well as downstream procedures. We present a workflow for gathering deeper process knowledge by a design of experiment approach for improved IGF1 IB formation in relation to impurity concentration. An IB expression maximum of 19.8 mgIGF1·gDCW-1 was found at pH 6.5, 37 °C and an IPTG induction of >45 µmol gDCW-1 for 12 h. Subsequently, three refolding buffers were tested together with a nonwoven anion exchange adsorber filter module. Knowledge-based buffer selection enabled high impurity log reduction values (LRVEndotoxin = 4.9; LRVDNA = 4.8, LRVHCP = 0.1-1) as well as chromatography column guarding potential by using those adsorptive matrices. Furthermore, adsorptive filtration followed by tangential flow filtration proved to be a promising alternative for product concentration.

Adsorptive filtration: A case study for early impurity reduction in an Escherichia coli production process.

Primary recovery of intracellular products from Escherichia coli requires cell disruption which leads to a massive release of process-related impurities burdening subsequent downstream process (DSP) unit operations. Especially, DNA and endotoxins challenge purification operations due to their size and concentrations. Consequently, an early reduction in impurities will not only simplify the production process but also increase robustness while alleviating the workload afterward. In the present work, we studied the proof of concept whether a nonwoven anion exchange filter material decreases soluble impurities immediately at the clarification step of E. coli DSP. In a first attempt, endotoxin burden was reduced by 4.6-fold and the DNA concentration by 3.6-fold compared to conventional depth filtration. A design of experiment for the adsorptive filtration approach was carried out to analyze the influence of different critical process parameters (CPPs) on impurity reduction. We showed that depending on the CPPs chosen, a DNA lowering of more than 3 log values, an endotoxin decrease of approximately 7 logs, and a minor HCP clearance of at least 0.3 logs could be achieved. Thus, we further revealed a chromatography column protecting effect when using adsorptive filtration beforehand.

Selective Targeting of a Disease-Related Conformational Isoform of Macrophage Migration Inhibitory Factor Ameliorates Inflammatory Conditions.

Macrophage migration inhibitory factor (MIF), a proinflammatory cytokine and counterregulator of glucocorticoids, is a potential therapeutic target. MIF is markedly different from other cytokines because it is constitutively expressed, stored in the cytoplasm, and present in the circulation of healthy subjects. Thus, the concept of targeting MIF for therapeutic intervention is challenging because of the need to neutralize a ubiquitous protein. In this article, we report that MIF occurs in two redox-dependent conformational isoforms. We show that one of the two isoforms of MIF, that is, oxidized MIF (oxMIF), is specifically recognized by three mAbs directed against MIF. Surprisingly, oxMIF is selectively expressed in the plasma and on the cell surface of immune cells of patients with different inflammatory diseases. In patients with acute infections or chronic inflammation, oxMIF expression correlated with inflammatory flare-ups. In addition, anti-oxMIF mAbs alleviated disease severity in mouse models of acute and chronic enterocolitis and improved, in synergy with glucocorticoids, renal function in a rat model of crescentic glomerulonephritis. We conclude that oxMIF represents the disease-related isoform of MIF; oxMIF is therefore a new diagnostic marker for inflammation and a relevant target for anti-inflammatory therapy.

Investigations into cytotoxic effects of the herbal preparation Abnormal Savda Munziq.

OBJECTIVE: To evaluate the effects of Abnormal Savda Munziq (ASMq), a traditional herbal medicine, for the prevention and treatment of human diseases, e.g. bowel cancer. METHODS: The parameters total polyphenol content, cell proliferation and DNA-damage as well as RNA and protein-oxidation were analysed in vitro. Besides, the expressions of miRNA and tumor suppressor genes as well as cellular senescence were evaluated. RESULTS: ASMq had a high polyphenol content and induced damage to proteins, RNA as well as to DNA, which is correlated with its cytotoxicity. Furthermore ASMq up-regulated the tumor suppressor genes p21, p53 and p16 and down-regulated the micro-RNAs hsa-mir-17 and hsa-mir-106b. In addition cellular growth arrest and SA-ß-gal-staining were induced. CONCLUSION: ASMq has the ability to induce DNA damage and cellular senescence, which are double-edged mechanisms in fighting cancer, as they might also have harmful side effects.

High levels of oncomiR-21 contribute to the senescence-induced growth arrest in normal human cells and its knock-down increases the replicative lifespan.

Cellular senescence of normal human cells has by now far exceeded its initial role as a model system for aging research. Many reports show the accumulation of senescent cells in vivo, their effect on their microenvironment and its double-edged role as tumour suppressor and promoter. Importantly, removal of senescent cells delays the onset of age-associated diseases in mouse model systems. To characterize the role of miRNAs in cellular senescence of endothelial cells, we performed miRNA arrays from HUVECs of five different donors. Twelve miRNAs, comprising hsa-miR-23a, hsa-miR-23b, hsa-miR-24, hsa-miR-27a, hsa-miR-29a, hsa-miR-31, hsa-miR-100, hsa-miR-193a, hsa-miR-221, hsa-miR-222 and hsa-let-7i are consistently up-regulated in replicatively senescent cells. Surprisingly, also miR-21 was found up-regulated by replicative and stress-induced senescence, despite being described as oncogenic. Transfection of early passage endothelial cells with miR-21 resulted in lower angiogenesis, and less cell proliferation mirrored by up-regulation of p21(CIP1) and down-regulation of CDK2. These two cell-cycle regulators are indirectly regulated by miR-21 via its validated direct targets NFIB (Nuclear factor 1 B-type), a transcriptional inhibitor of p21(CIP) (1) , and CDC25A, which regulates CDK2 activity by dephosphorylation. Knock-down of either NFIB or CDC25A shows a phenocopy of over-expressing miR-21 in regard to cell-cycle arrest. Finally, miR-21 over-epxression reduces the replicative lifespan, while stable knock-down by sponges extends the replicative lifespan of endothelial cells. Therefore, we propose that miR-21 is the first miRNA that upon its knock-down extends the replicative lifespan of normal human cells.

Abnormal Savda syndrome: long-term consequences of emotional and physical stress on endocrine and immune activities in an animal model.

OBJECTIVE: To investigate the relationship between emotional status, cold-dry environment and long-term immune responses to the stressors, and the potential pathological mechanisms between causative factors of abnormal Savda syndrome (ASS) and the susceptibility to disease; thus to clarify the ASS, and secondly to identify the optimal ASS animal model for further studies on traditional Uighur therapeutical formulations. METHODS: Sixty mice were randomly and equally divided into 4 groups: control and 3 stress groups. The cold-dry environment was applied by keeping the mice in a climatic chamber. The emotional stress was induced by the application of the repeated electric foot-shocks in the electric foot-shock apparatus. The mice of the combined stress group underwent the repeated electric foot-shock treatment before being housed in the climatic chamber. The experimental routine was repeated for 21 days. In order to look into endocrine and immune stress responses, ELISA was used to determine the serum levels of the hormones corticotropin-releasing hormone (CRH), adrenocorticotropic hormone (ACTH), Beta-endorphin (ß-END) and corticosterone (CORT), of the cytokines interleukin 2 (IL-2), interleukin 6 (IL-6), interferon-gamma (INF-γ) and tumor necrosis factor-alpha (TNF-α), and of the immunoglobulins immunoglobulin A (IgA), immunoglobulin M (IgM) and immunoglobulin G (IgG). Lymphocyte subsets were analyzed in duplicate in order to determine differences in the T cell ratio. RESULTS: In the cold-dry environment group, the serum levels of CRH, ACTH and CORT were significantly higher than those of the control group, whereas serum ß-END was not found significantly different. In both the repeated electric foot-shock group as well as in the combined stress group the serum levels of CRH, ACTH, ß-END and CORT were significantly higher. Compared to the control animals, the serum concentration of INF-γ was significantly lower in all three different stress groups. The serum level of IL-2 was decreased in the combined stress group whereas the serum TNF-α level was significantly higher. The serum IgG level was significantly higher in all three stress groups, whereas the IgA level was lower in both chronic electric foot-shock group and combined stress group. The IgM level was found significantly higher in the combined stress group only. The percentage of CD4(+) cells in peripheral blood was dramatically decreased in mice exposed to colddry environment, chronic electric foot-shock and combined stress, whereas the percentage of the CD8(+) subset was not significantly different. The CD4(+)/CD8(+) ratios were markedly lower in both cold-dry environment group and combined stress group. CONCLUSIONS: Combined stress can cause hyperactivity of the HPA axis, and an imbalance in the Th1/Th2 cell subset may contribute to illustrate the partial pathological mechanisms of ASS. This study identified this animal model of a combination of physical and emotional stress as an optimal model for further studies on ASS and relative therapies.

HIV-1 induces telomerase activity in monocyte-derived macrophages, possibly safeguarding one of its reservoirs.

Monocyte-derived macrophages (MDM) are widely distributed in all tissues and organs, including the central nervous system, where they represent the main part of HIV-infected cells. In contrast to activated CD4(+) T lymphocytes, MDM are resistant to cytopathic effects and survive HIV infection for a long period of time. The molecular mechanisms of how HIV is able to persist in macrophages are not fully elucidated yet. In this context, we have studied the effect of in vitro HIV-1 infection on telomerase activity (TA), telomere length, and DNA damage. Infection resulted in a significant induction of TA. This increase was directly proportional to the efficacy of HIV infection and was found in both nuclear and cytoplasmic extracts, while neither UV light-inactivated HIV nor exogenous addition of the viral protein Tat or gp120 affected TA. Furthermore, TA was not modified during monocyte-macrophage differentiation, MDM activation, or infection with vaccinia virus. HIV infection did not affect telomere length. However, HIV-infected MDM showed less DNA damage after oxidative stress than noninfected MDM, and this resistance was also increased by overexpressing telomerase alone. Taken together, our results suggest that HIV induces TA in MDM and that this induction might contribute to cellular protection against oxidative stress, which could be considered a viral strategy to make macrophages better suited as longer-lived, more resistant viral reservoirs. In the light of the clinical development of telomerase inhibitors as anticancer therapeutics, inhibition of TA in HIV-infected macrophages might also represent a novel therapeutic target against viral reservoirs.

miR-17, miR-19b, miR-20a, and miR-106a are down-regulated in human aging.

Aging is a multifactorial process where deterioration of body functions is driven by stochastic damage while counteracted by distinct genetically encoded repair systems. To better understand the genetic component of aging, many studies have addressed the gene and protein expression profiles of various aging model systems engaging different organisms from yeast to human. The recently identified small non-coding miRNAs are potent post-transcriptional regulators that can modify the expression of up to several hundred target genes per single miRNA, similar to transcription factors. Increasing evidence shows that miRNAs contribute to the regulation of most if not all important physiological processes, including aging. However, so far the contribution of miRNAs to age-related and senescence-related changes in gene expression remains elusive. To address this question, we have selected four replicative cell aging models including endothelial cells, replicated CD8(+) T cells, renal proximal tubular epithelial cells, and skin fibroblasts. Further included were three organismal aging models including foreskin, mesenchymal stem cells, and CD8(+) T cell populations from old and young donors. Using locked nucleic acid-based miRNA microarrays, we identified four commonly regulated miRNAs, miR-17 down-regulated in all seven; miR-19b and miR-20a, down-regulated in six models; and miR-106a down-regulated in five models. Decrease in these miRNAs correlated with increased transcript levels of some established target genes, especially the cdk inhibitor p21/CDKN1A. These results establish miRNAs as novel markers of cell aging in humans.